CN108653256B

CN108653256B - Composite nano diamond medicine and preparation method and application thereof

Info

Publication number: CN108653256B
Application number: CN201810585170.9A
Authority: CN
Inventors: 李英奇; 杜祥斌; 赵文静
Original assignee: Shanxi University
Current assignee: Shanxi University
Priority date: 2018-04-17
Filing date: 2018-06-08
Publication date: 2020-09-29
Anticipated expiration: 2038-06-08
Also published as: CN108653256A

Abstract

The invention provides a composite nano diamond medicine and a preparation method and application thereof. The drug takes Nanodiamond (ND) as a carrier, and Mitoxantrone (MTO) and adriamycin (DOXorubicin, DOX) are sequentially loaded on the surface of ND in a physical adsorption mode in the presence of sodium citrate to obtain the composite nano drug (ND/MTO/DOX). The change condition of the mitochondrial membrane potential of the cells is detected by a JC-1 method, and the maximum degree of mitochondrial membrane potential reduction caused by the composite nano-drug is found; the MTT method proves that the killing power to cancer cells is improved to a great extent by the synergistic effect of MTO and DOX, and the composite nano-drug has the characteristic of slow-release drug; the confocal result shows that the ND/MTO/DOX composite nano-drug is mainly positioned in lysosome, and the drug does not migrate to nucleus with time, and we speculate that the system may cause the increase of the permeability of the lysosome, so that a large amount of hydrolase is released, and the apoptosis of cells is promoted. The composite nano diamond medicine can be applied to preparing anti-tumor medicines.

Description

Composite nano diamond medicine and preparation method and application thereof

Technical Field

The invention relates to a nano-drug, in particular to a nano-diamond multiple drug simultaneously loaded with two anticancer drugs, and specifically relates to a nano-diamond/mitoxantrone/adriamycin composite nano-diamond drug and a preparation method and application thereof.

Background

The problems of food safety and environmental pollution are becoming more and more serious in the current society, and the incidence rate of cancer is rising year by year, and the cancer becomes one of the most important human killers. At present, the cancer treatment means mainly comprise: three general methods of surgery, radiation and chemical drug therapy. Among them, chemotherapy is widely used in clinical applications, such as adriamycin, methotrexate, and mitoxantrone, because of its simple operation. Chemotherapy is effective to some extent, but its nonspecific biodistribution in normal tissues and effects on healthy rapidly dividing cells result in severe toxic side effects in patients. In addition, chemotherapy drugs have many defects of poor solubility, rapid degradation in vivo, short blood circulation time and the like, so that the design of a high-efficiency and low-toxicity drug targeting delivery system for treating cancer is urgently needed.

With the knowledge of tumor microenvironment and the rapid development of nanotechnology in recent years, nanomaterials have become the "research pet" of scientists. Among the numerous nanomaterials, Nanodiamond (ND) has been widely used for targeted delivery of drugs and genes due to its advantages of good biocompatibility, easy surface modification, and large specific surface area. Anthracycline-based chemotherapeutic drugs, such as doxorubicin, epirubicin, daunorubicin, can be physically adsorbed on the ND surface by electrostatic adsorption, pi-pi interaction, van der waals forces, hydrogen bonding, and the like. Even though nanodiamond drug delivery systems are particularly free drugs in many respects, tumors remain difficult to eliminate and are prone to relapse due to the fact that the nanomaterial system carries only one chemotherapeutic drug. Therefore, there is a need for a new system of nanodiamond that can simultaneously carry multiple molecules of anticancer drugs and can synergistically treat tumors.

Mitoxantrone (MTO) is an artificially synthesized anthracycline antitumor drug, is an anticancer drug widely used clinically, shows a high curative effect in breast cancer, acute leukemia and lymphoma, and can be loaded on ND by physical adsorption. Based on the structural similarity of adriamycin and mitoxantrone, multiple nano-drugs are prepared by physically adsorbing chemotherapy drugs mitoxantrone and adriamycin by carboxylated nano-diamonds under the action of sodium citrate, and the anti-tumor activity of the drugs is researched.

Disclosure of Invention

The invention aims to provide a nano diamond drug simultaneously loaded with two chemotherapeutic drugs, a preparation method thereof and application of the drug in tumor resistance.

The invention provides a preparation method of a nano diamond drug simultaneously loaded with two chemotherapeutic drugs (mitoxantrone and adriamycin), which comprises the following steps:

according to the mass ratio of 7-10: 1.5-3: 1, weighing ND, MTO and DOX, firstly ultrasonically dispersing ND in a sodium citrate solution, adding MTO after 30min, stirring for 0.5-1.5h at room temperature, centrifuging, washing with distilled water until the supernatant is colorless, collecting the supernatant, and recording the volume of the liquid to obtain the ND/MTO nano-drug; and ultrasonically dispersing the ND/MTO nano-drug in a sodium citrate solution for 30min, adding DOX, stirring at room temperature for 0.5-1.5h, centrifuging, washing with distilled water until the supernatant is colorless, collecting the supernatant, recording the liquid volume, and drying the obtained ND/MTO/DOX composite nano-drug in a vacuum freeze dryer for later use.

The mass ratio of ND, MTO and DOX is preferably 8.3: 2: 1.

the concentration of the sodium citrate solution was 1.0M.

The stirring time at room temperature is preferably 1 h.

The prepared composite nano-diamond medicament can be applied to synergistic antitumor.

Compared with the prior art, the invention has the beneficial effects that: the nano diamond material selected by the invention has the advantages of good biocompatibility, easy surface modification, large specific surface area and the like; mitoxantrone and adriamycin are two antitumor drugs widely applied to clinic, show higher curative effect in treating breast cancer and lymphoma, and can be loaded on ND through physical adsorption; due to the fact that the adriamycin and the mitoxantrone are similar in structure, the adriamycin and the mitoxantrone are conveniently loaded on the surface of the ND in a physical adsorption mode under the action of the sodium citrate, and the preparation method is simple and time-saving. The anticancer effect of the prepared composite nano-diamond medicament is superior to that of a single nano-diamond medicament.

Earlier researches show that after the nanodiamond medicine carrying system is taken up by cells, the medicine is dissociated and enters cell nucleuses so as to induce cell apoptosis, and the ND/MTO/DOX composite nano medicine new system prepared by the application can release a large amount of lysosome hydrolase in a mode of changing lysosome permeability, so that the aim of inhibiting tumor growth is finally achieved.

The JC-1 method is used for detecting the mitochondrial membrane potential change condition of the composite nano-drug in human liver cancer cells (HepG2), the nano-diamond is found to have almost no influence on the composite nano-drug, and compared with a free drug, the composite nano-drug causes the maximum reduction range of the mitochondrial membrane potential, which implies that the nano-diamond is an excellent carrier and has low toxicity, and the synthesized composite nano-drug has better curative effect on the treatment of cancers; the laser confocal result shows that the ND/MTO/DOX composite nano-drug is mainly present in cytoplasm and is positioned in lysosome; the activity of the composite nano-drug on HepG2 cells is detected by an MTT method, which shows that the ND/MTO/DOX composite nano-drug has anticancer selectivity, and simultaneously, the synergistic effect of MTO and DOX is utilized, so that the curative effect of the composite nano-drug system is superior to that of a single nano-drug, the lethality on cancer cells is improved to a great extent, and the composite nano-drug has the characteristic of slow release of the drug, so that the ND/MTO/DOX composite nano-diamond drug can be applied to the preparation of antitumor drugs.

Drawings

FIG. 1 Infrared Spectroscopy of various nanoparticles

FIG. 2 scanning electron micrographs of various nanoparticles

FIG. 3 JC-1 staining method for detecting influence of ND/MTO/DOX composite nano-drug on mitochondrial membrane potential

FIG. 4 ND/MTO/DOX composite nano-drug anticancer selectivity

FIG. 5 Effect of different drugs on the Activity of HepG2 cells cultured in vitro (24h)

FIG. 6 Effect of different drugs on the Activity of HepG2 cells cultured in vitro (48h)

FIG. 7 Effect of nanodiamonds on the Activity of HepG2 cells cultured in vitro (48h)

FIG. 8 intracellular imaging of ND/MTO/DOX composite nano-drug

FIG. 9 intracellular localization diagram of ND/MTO/DOX composite nano-drug

Detailed Description

The materials used in the examples are as follows:

nanodiamond (ND, element six, diameter about 140nm, Ireland).

Adriamycin (DOX) is doxorubicin hydrochloride, a moleculeFormula is C₂₇H₂₉NO₁₁HCl, molecular weight 579.99, Shanxipu pharmaceutical Co., Ltd, specification C₂₇H₂₉NO₁₁HCl calculated as 10 mg.

Mitoxantrone (MTO) molecular formula C₂₂H₃₀Cl₂N₄O₆Molecular weight of 517.4, Shanghai Biotech Co., Ltd., specification C₂₂H₃₀Cl₂N₄O₆HCl calculated as 1 g.

Sodium citrate dihydrate (C)₆H₅Na₃O₇·2H₂O, molecular weight 294.10) was produced by Tianjin Ministry of peak chemistry.

Example 1

A method for preparing a nanodiamond/mitoxantrone/adriamycin (ND/MTO/DOX) composite nanodiamond medicament comprises the following steps:

weighing 1mg of ND, ultrasonically dispersing the ND in a sodium citrate (Na3Cit, 1.0M, 1mL) solution, adding 240 mu g of MTO after 30min, stirring for 1h at room temperature, centrifuging, washing with distilled water until the supernatant is colorless, collecting the supernatant, and recording the volume of the liquid to obtain the ND/MTO nano-drug. Then, the ND/MTO nano-drug is ultrasonically dispersed in a sodium citrate solution, after 30min, 120 mu g of DOX is added, the mixture is stirred for 1h at room temperature, the centrifugation is carried out, the mixture is washed by distilled water until the supernatant is colorless, the supernatant is collected, the liquid volume is recorded, and the obtained nano-diamond/mitoxantrone/adriamycin (ND/MTO/DOX) composite nano-diamond drug is placed in a vacuum freeze drier for drying for later use.

By measuring the absorbance of DOX at 480nm (DOX)_480nm＝11500cm^-1·mol^-1·L^-1) The absorbance of MTO at 609nm was calculated from the respective standard curves, and the respective contents of the supernatants were calculated, whereby it was found that the respective adsorption amounts of MTO and DOX on ND were 119.2. + -. 3.0. mu.g/mg and 60.1. + -. 3.0. mu.g/mg, respectively.

Example 2

Characterization of the Infrared Spectroscopy

By means of an infrared spectrometer, the surface chemical properties of the ND/MTO/DOX composite nano-drug can be characterized and determinedWhether MTO and DOX are adsorbed on the ND surface or not and whether the ND/MTO/DOX composite nano-medicament contains Na or not₃Cit. Taking a small amount of dried nanoparticles, mixing with KBr, tabletting, measuring the respective infrared spectrogram by using an infrared spectrometer, and analyzing the spectrogram.

FIG. 1 is an infrared spectrum of various materials. As shown in FIG. 1(A), in the infrared spectrum of ND, it can be seen that the surface thereof is rich in carboxyl groups, indicating that ND is successfully oxidized by strong acid; in the infrared spectrogram of ND/MTO/DOX composite nano-drug, the characteristic absorption peak marked by the frame and Na in the figure 1(B)₃The characteristic absorption peaks of the Cit infrared spectrogram (frame mark) are consistent, which indicates that Na exists in the ND/MTO/DOX nano-drug₃Cit. Meanwhile, in an ND/MTO/DOX composite nano-drug infrared spectrogram, the compound nano-drug not only contains a characteristic peak (1094 cm) from a benzene ring in a DOX molecule^-1: C-O contraction vibration peak; 1409cm^-1: in-plane deformation vibration peak of hydroxyl group), and C-O stretching vibration peak (1214 cm) of phenolic hydroxyl group in MTO molecule^-1) And the out-of-plane bending vibration peak of C-H (824 cm)^-1) The result proves that the ND/MTO/DOX composite nano-medicament contains MTO, DOX and Na₃Cit, the nano-drug is successfully prepared.

Example 3

In order to observe the morphology of various nano materials, a Scanning Electron Microscope (SEM) is adopted to characterize ND, ND/MTO and ND/MTO/DOX nano drugs. And (3) taking a small amount of dried nano particles to be placed on conductive adhesive, carrying out sample adhesion, spraying gold, and carrying out machine detection.

FIG. 2 is a Scanning Electron Microscope (SEM) image of ND, ND/MTO and ND/MTO/DOX nano-drug, from which it can be seen that there are clearly stacked "cloud clusters" (indicated by arrows) on the surface and more on the surface of ND/MTO/DOX compared to ND. This is probably because the existence of sodium citrate induces the aggregation of MTO and DOX, and pi-pi stacking phenomenon exists among ND, MTO and DOX, and the experiment proves the successful synthesis of the nano-drug again.

Example 4

The decrease in mitochondrial membrane potential is a characteristic marker of early apoptosis of cells, which is irreversible if the breakdown of the mitochondrial transmembrane potential occurs. By adopting a mitochondrial membrane potential detection kit (JC-1), the mitochondrial membrane potential change condition of cells can be rapidly and sensitively detected. The membrane potential of mitochondria of apoptotic cells is reduced or deleted, so that the fluorescent probe JC-1 exists in a monomer form, and is excited at 488nm to show green fluorescence; the normal and healthy mitochondria have high membrane potential, JC-1 exists in a polymer form, red fluorescence is presented, and the membrane potential reduction degree of the mitochondria can be evaluated by detecting the conversion degree from the red fluorescence to the green fluorescence.

HepG2 cells grown in log phase (density: 1.8 × 10)⁵One dish) were inoculated in a 35mm petri dish and placed in an incubator (37 ℃, 5% CO)₂) Culturing until the cells are completely attached, removing the old solution, adding culture solution containing ND (2.8. mu.g/mL), DOX (0.5. mu.g/mL), MTO (0.5. mu.g/mL), DOX + MTO (0.5. mu.g/mL) and ND/MTO/DOX (ND: 2.8. mu.g/mL, DOX + MTO: 0.5. mu.g/mL) respectively, incubating for 24h, removing the old solution, washing with cold PBS for 2-3 times, collecting the cells with trypsin, pre-machine centrifuging, resuspending the cells with vortex mixed JC-1 working solution (500. mu.L 1 × Incubation Buffer + 1. mu.LJC-1), incubating for 15-20min in a cell culture box, washing with 1 × ubbuffer for 1-2 times, removing the unabsorbed JC-1 to avoid interference, and detecting on-machine by adopting a cytometer (excitation: 488 nm; red fluorescence emission: 580/590 nm; green fluorescence emission: 510/527nm) to represent the change of the fluorescence intensity of the red-emitting membrane of the red corpuscles.

FIG. 3 shows the effect of various materials on mitochondrial membrane potential, FL1 is used to represent the intensity of green light, FL2 is used to represent the intensity of red light, and thus the early killing power of the drug on cancer cells can be judged by comparing FL1/FL2 values. It is understood from the figure that FL1/FL2 values of ND, DOX, MTO: DOX and ND/MTO/DOX are 0.06%, 4.33%, 33.79%, 30.39% and 54.41%, respectively, from which ND particles have almost no effect on mitochondrial membrane potential of cells, and ND/MTO/DOX makes the mitochondrial membrane potential decrease the most extent compared to the free drug, suggesting that ND is an excellent carrier and that the synthesized composite nano-drug has a better efficacy for cancer treatment.

Example 5

To search for anticancer selectivity of ND/MTO/DOX nano-drug, we used MTT method to study the difference of toxicity effects of drug concentration and action time on HepG2 cancer cell and rat adrenal gland chromaffin tumor cell (PC-12) cell lines, HepG2 cell and PC-12 cell (cell density per well: 5.0 × 10) in logarithmic growth phase³Respectively) were inoculated on a 96-well plate, and 16h after the plate was attached to the wall, a series of nano-drugs containing ND/MTO/DOX at different concentrations (total concentration of drug: 0.5, 1, 2 and 4. mu.g/mL), 6 multiple wells were set with untreated cells as a control, and after 24h and 48h, 5mg/mL MTT (20. mu.L) was added to each well, and after 4-6h in an incubator, the old solution was discarded, followed by addition of 150. mu.L DMSO, shaking for 10min, and measurement was performed using an enzyme linked immunosorbent assay.

FIG. 4 is a graph showing the difference between the ND/MTO/DOX nano-drug system acting on HepG2 cancer cell and mouse adrenal gland chromaffin tumor cell (PC-12) cell lines. We found that the toxicity of the nano-drug to PC-12 was substantially less than that to HepG2 cancer cells, regardless of the time period of 24h (FIG. 4A) or 48h (FIG. 4B) or at any concentration, and the difference between the two was more significant at higher concentrations. The nano drug-loaded system is shown to have anticancer selectivity, so that the biological effects of the nano drug-loaded system and HepG2 cancer cells can be deeply researched.

Example 6

Influence of ND/MTO/DOX nano-drugs with different concentrations on activity of human liver cancer cells (HepG2)

In order to quantitatively explore the influence of ND/MTO/DOX nano-drugs on the activity of human liver cancer cells (HepG2), the influence of drugs with different concentrations and different treatment times on the growth condition of the cells is researched by an MTT method, and the specific method is that HepG2 cells (cell density per well: 5.0 × 10) in logarithmic growth phase are treated³Respectively) inoculating the cells to a 96-well plate, after 16h of adherence, respectively adding 10% FBS/DMEM culture medium containing different concentrations of DOX, MTO, DOX + MTO, ND/MTO, ND/DOX and ND/MTO/DOX to prepare 200 mu L of culture solution, taking untreated cells as a control group, setting 6 multiple wells, respectively culturing for 24h and 48h, adding 5mg/mL MTT (20 mu L) into each well, placing in an incubator for 4-6h, then removing old solution, and then adding 150 h_μL DMSO, shaking for 10min, and detecting with enzyme-linked immunosorbent assay (ELISA).

FIGS. 5 and 6 are IC's for different drugs₅₀Curves (24h in fig. 5 and 48h in fig. 6). As shown in the figure, under the same concentration, the lethality of the free MTO-DOX composite drug to HepG2 cells is better than that of MTO and DOX, which proves the synergistic effect of the composite drug combination therapy, and the same condition is applicable to ND/MTO/DOX, ND/DOX and ND/MTO, namely the former has better anti-cancer effect than the latter two; and by comparing the six curves, the slope of the curves of the nano-drug systems (ND/MTO/DOX, ND/DOX and ND/MTO) is relatively gentle compared with the three free drugs, which shows that the three nano-drug systems have a slow release effect on the release of the drugs. Meanwhile, the biocompatibility of the nano-carrier ND is tested (figure 7), as shown in the figure, under the condition of different concentrations, the nano-carrier ND hardly influences the activity of HepG2 cells within 48 hours, the cell survival rate is over 90 percent, and the nano-carrier ND is good in biocompatibility and can be used as a safe nano-carrier for effective drug delivery.

Example 7

Subsequently, we compare the IC of each group of nano-drugs with FIG. 5 and FIG. 6₅₀Values were counted and the combination index CL was used to evaluate the effectiveness of different drugs in treating HepG2 cells at different times. Combination index CL less than 1, CL equal to 1 and CL greater than 1 are expressed as synergistic effect of the combination, respectively, additive and antagonistic therapies the combination index (CL) was used to assess whether the binding of MTO to DOX had a synergistic effect on HepG2 cells, and the CL values were calculated according to equations (1.1) and (1.2):

Dose＝Dose₁+Dose₂（1.1)

wherein Dose is x% of total Dose of drug 1 and drug 2 in the composition₁And Dose₂The doses of drug 1 and drug 2 that inhibited cells by x% in the combination are expressed separately (assuming that both drugs are released at a molar ratio MTO: DOX ═ 2: 1).

Wherein Dose₁And Dose₂Represents the x% cytostatic dose of drug 1 and drug 2, respectively, in the combination, whereas Dosex₁And Dosex₂Represent the doses of drug 1 and drug 2, respectively, that inhibited x% of the cells in the corresponding monotherapy. See tables 1 and 2 for details.

TABLE 1 IC Effect of different classes of drugs on HepG2 cells₅₀Value (half inhibitory concentration) and corresponding combination index (24h)

TABLE 2 IC Effect of different drugs on HepG2 cells₅₀Value (half inhibitory concentration) and corresponding combination index (48h)

As can be seen from tables 1 and 2, the anticancer effect of free drug DOX is not as good as that of free MTO at 24h (Table 1) or 48h (Table 2); at 24h, the anticancer effect of ND/DOX is better than that of ND/MTO, which is probably because the pi-pi interaction between MTO is larger than that between DOX, so that the release rate of MTO is smaller than that of DOX, and at 48h, the anticancer effect of ND/MTO is found to be more excellent, which is presumably because MTO is more favorably released along with the prolonging of time and the release amount is enough to cause apoptosis, so that the anticancer effect of ND/MTO is higher than that of DOX; meanwhile, at 24h, compared with single-drug therapy, the combination index CL of the free MTO-DOX compound drug and the ND/MTO/DOX compound nano-drug is less than 1, and the CL values are reduced along with the time increase to 48h, which fully indicates that the combined administration of MTO and DOX can increase the lethality rate to HepG2 cells, and the synergistic effect is more obvious along with the time increase.

Example 8

To investigate whether ND/MTO/DOX nanomedicines could enter living cells, we used nitrogen-doped red Fluorescent Nanodiamond (FND) instead of non-fluorescent Nanodiamond (ND) HepG2 cells (density: 2.0 × 10)⁵Individual cells/dish) seeded on a coverslipper at the bottom35mm petri dish. After 16h of adherence, the old solution is discarded, and the cells are incubated for 15h, 19h and 24h by using a culture solution containing FND/MTO/DOX (FMD) composite nano-drugs. After timing was complete, the cells were washed 3 times with PBS and free FMD was removed. Subsequently, after fixing the cells with 4% paraformaldehyde at room temperature, nuclei were stained with Hoeschst 33258. All images were obtained using a confocal laser microscope (FND: excitation 543 nm; DOX: excitation 488 nm; Hoeschst 3325: excitation 405 nm).

Fig. 8 shows confocal fluorescence images of HepG2 cells treated with FMD at different time points. It can be seen from the figure that after FMD treatment, red fluorescence and green fluorescence can be observed, and that some orange-yellow fluorescence in the cytoplasm gradually increases with time, which shows co-localization of red FND nanopharmaceuticals with green fluorescing doxorubicin, and no DOX entry into the nucleus is found with time up to 24 h.

Example 9

To further track the specific location of ND/MTO/DOX complex nanopharmaceuticals in cells, we employed red lysosomal probes. The method comprises the following specific steps: after incubation of adherent HepG2 cells with ND/MTO/DOX (DOX + MTO: 6. mu.g/mL) for 15h, the cells were washed 2-3 times with ice-cold PBS (pH 7.4) to remove free ND/MTO/DOX. Then a red lysosomal probe (Lyo-Tracker probe) was added and incubation continued for 5-10 min. After washing the cells 3 times with ice-cold PBS (pH 7.4), the cells were fixed with 4% paraformaldehyde for 8 min. Finally, the cells were observed using a confocal laser scanning microscope (Lyo-Tracker probe, excitation: 568nm) and photographed.

FIG. 9 is an image of cells incubated with ND/MTO/DOX composite nano-drug for 15 h. In fig. 9A we can observe red and green fluorescence, respectively, and in the overlay some orange fluorescence in the cytoplasm can be observed, which is a high co-localization showing green DOX with lysosomes labeled red. The co-localization coefficient shown in fig. 9B is 0.70, and in general, when the co-localization coefficient is close to or greater than 0.5, it can be considered as a good co-localization index. Therefore, we speculate that the ND/MTO/DOX composite nano-drug is trapped in lysosomes after following the cycle process of endocytosis, so that the permeability of lysosomes is increased, a large amount of lysosome hydrolase is released, and apoptosis is promoted.

Claims

1. A method for preparing a nanodiamond/mitoxantrone/adriamycin (ND/MTO/DOX) composite nanodiamond medicament is characterized by comprising the following steps:

according to the mass ratio of 8.3: 2:1, weighing ND, MTO and DOX, firstly ultrasonically dispersing ND in a sodium citrate solution, adding MTO after 30min, stirring for 0.5-1.5h at room temperature, centrifuging, washing with distilled water until the supernatant is colorless, collecting the supernatant, and recording the volume of the liquid to obtain the ND/MTO nano-drug; and ultrasonically dispersing the ND/MTO nano-drug in a sodium citrate solution for 30min, adding DOX, stirring at room temperature for 0.5-1.5h, centrifuging, washing with distilled water until the supernatant is colorless, collecting the supernatant, recording the liquid volume, and drying the obtained ND/MTO/DOX composite nano-diamond drug in a vacuum freeze dryer for later use.

2. The method of claim 1, wherein said sodium citrate solution is at a concentration of 1.0M.

3. The method of claim 1, wherein the stirring at room temperature is performed for 1 hour.

4. A composite nanodiamond medicament prepared according to any one of claims 1-3.

5. Use of the composite nanodiamond medicament prepared according to any one of claims 1-3 for the preparation of an anti-tumor medicament.